Capturing Enzyme-Loaded Diblock Copolymer Vesicles Using an Aldehyde-Functionalized Hydrophilic Polymer Brush

Abstract

Compared to lipids, block copolymer vesicles are potentially robust nanocontainers for enzymes owing to their enhanced chemical stability, particularly in challenging environments. Herein we report that cis-diol-functional diblock copolymer vesicles can be chemically adsorbed onto a hydrophilic aldehyde-functional polymer brush via acetal bond formation under mild conditions (pH 5.5, 20 °C). Quartz crystal microbalance studies indicated an adsorbed amount, Γ, of 158 mg m^-2 for vesicle adsorption onto such brushes, whereas negligible adsorption (Γ = 0.1 mg m^-2) was observed for a control experiment conducted using a cis-diol-functionalized brush. Scanning electron microscopy and ellipsometry studies indicated a mean surface coverage of around 30% at the brush surface, which suggests reasonably efficient chemical adsorption. Importantly, such vesicles can be conveniently loaded with a model enzyme (horseradish peroxidase, HRP) using an aqueous polymerization-induced self-assembly formulation. Moreover, the immobilized vesicles remained permeable toward small molecules while retaining their enzyme payload. The enzymatic activity of such HRP-loaded vesicles was demonstrated using a well-established colorimetric assay. In principle, this efficient vesicle-on-brush strategy can be applied to a wide range of enzymes and functional proteins for the design of next-generation immobilized nanoreactors for enzyme-mediated catalysis.

Scheme 1. Synthetic Route for the Preparation of HRP-Loaded cis-Diol-Functional PGEO5MA₁₉-PHPMA₅₀₀ (GO₁₉-H₅₀₀) Vesicles via RAFT Aqueous Dispersion Polymerization of HPMA at 37 °C Targeting 10% w/w Solids

An identical protocol was used to produce empty (enzyme-free) vesicles.

Figure 1

Representative ellipsometry data (solid lines) and fits (dashed) with schematic cartoons for the four interfacial structures and their corresponding ellipsometry models. Psi values are shown in blue, while delta values are shown in red. (A) Dry PAGEO5MA brush represented in the ellipsometry model by a single Cauchy layer with variable thickness (Cauchy parameters in the main text). (B) Hydrated PAGEO5MA brush represented by a single effective medium approximation (EMA) layer with a variable thickness and water content. (C) Dry vesicle-on-brush represented in the ellipsometry model by a single Cauchy layer with variable thickness (same Cauchy parameters as used for panel A). (D) Solvated vesicle-on-brush system, which required two EMA layers to adequately fit the data. Each layer comprised a mixture of polymer and water and the thickness and composition was allowed to vary in both layers.

Figure 2

(A) Normalized SEC data recorded for the GO₁₉ precursor (black curve), GO₁₉-H₅₀₀ diblock copolymer (red curve), and the GO₁₉-H₅₀₀ diblock copolymer prepared in the presence of HRP (green curve). (B) DLS data obtained for empty (red curve) and HRP-loaded (green curve) GO₁₉-H₅₀₀ vesicles. (C) Representative TEM images (uranyl formate stain) recorded for GO₁₉-H₅₀₀ vesicles (red border image—left) and HRP-loaded GO₁₉-H₅₀₀ vesicles (green border image—right).

Scheme 2. (i) Preparation of a cis-Diol-Functional PGEO5MA Precursor Brush via SI-ARGET ATRP Using Either a Planar Silicon Wafer or a Glass Slide, (ii) Selective Oxidation to Produce the Corresponding Aldehyde-Functional PAGEO5MA Brush, and (iii) Its Subsequent Exposure to Either GO₁₉-H₅₀₀ or HRP-GO₁₉-H₅₀₀ Vesicles to Produce Vesicle-On-Brush Systems

Figure 3

(A) Representative AFM images recorded for PGEO5MA brushes grown from patterned initiator films on planar glass slides for various polymerization times. (B) Line sections through AFM images of patterned brushes. (C) Increase in the dry brush thickness with polymerization time for PGEO5MA brushes grown from a planar silicon wafer and a glass slide. Dry brush thicknesses were determined by ellipsometry analysis for planar silicon wafers and from AFM images for glass slides (ellipsometry thickness data reproduced from Brotherton et al.).

Figure 4

Fluorescence microscopy images recorded for (A) a 1.0% w/w aqueous dispersion of FITC-labeled GO₁₉H₅₀₀ vesicles in solution and (B) the vesicle-on-brush system: FITC-labeled GO₁₉H₅₀₀ vesicles adsorbed at pH 5.5 on a 116 nm PAGEO5MA brush grown from a patterned initiator film on a planar silicon wafer.

Figure 5

(A) Representative SEM images recorded for PAGEO5MA brush (A) prior to vesicle adsorption and (B) after chemical adsorption via acetal chemistry. (C) SEM image recorded for a non-reactive cis-diol-functionalized PGEO5MA brush after exposure to HRP-loaded GO₁₉-H₅₀₀ vesicles (control experiment).

Figure 6

Change in frequency of the third overtone, Δf₃, over time at 25 °C for a QCM silica sensor coated with either a 43 nm cis-diol-functionalized PGEO5MA brush (blue) or a 41 nm aldehyde-functionalized PAGEO5MA brush (green) after exposure to an aqueous dispersion of HRP-loaded vesicles (1.0 wt %) in PB solution at pH 5.5. Using the Sauerbrey equation, the corresponding adsorbed amount, Γ, was calculated to be 0.1 mg m^–2 for the cis-diol-functionalized PGEO5MA brush and 158 mg m^–2 for the aldehyde-functionalized PAGEO5MA brush.

Figure 7

(A) HRP-catalyzed oxidation of DMB generates a red dimer product inside HRP-loaded GO₁₉-H₅₀₀ vesicles. (B) Enzymatic activity of free HRP diluted in 100 mM PB solution at pH 5.5 at various enzyme concentrations. (C) Enzymatic activity of purified HRP-loaded GO₁₉-H₅₀₀ vesicles (green curve) compared to that observed for empty GO₁₉-H₅₀₀ vesicles (red curve). (D) Enzymatic activity of HRP-loaded GO₁₉-H₅₀₀ vesicles chemically adsorbed onto an aldehyde-functional PAGEO5MA brush (blue curve) relative to that observed for three control experiments (red, green, and black curves).